甲型流感病毒樣顆粒疫苗的初步研究
發(fā)布時(shí)間：2018-08-14 10:49
【摘要】：流感是世界范圍內(nèi)廣泛流行的呼吸系統(tǒng)傳染病,每年都造成大量的住院及死亡病例。截至2010年7月18日,爆發(fā)于墨西哥的甲型H1N1流感已在全球214個(gè)國(guó)家造成18,336人死亡;自2003年以來(lái),H5N1高致病性禽流感病毒已在世界范圍內(nèi)造成310人死亡,且死亡率高達(dá)59%。疫苗是防控流感最為有效的手段。流感疫苗的發(fā)展經(jīng)歷了滅活疫苗、裂解疫苗、減毒活疫苗、亞單位疫苗等階段,季節(jié)性流感裂解疫苗雖然能在人群中產(chǎn)生有效保護(hù)作用,但是依然存在許多問題。首先,傳統(tǒng)流感疫苗的生產(chǎn)工藝依賴于雞胚,難以應(yīng)對(duì)流感大規(guī)模爆發(fā)對(duì)疫苗的需求,且雞胚卵清蛋白易引起過敏,至今還沒有針對(duì)老年人、兒童及特殊人群理想的流感疫苗,因此現(xiàn)有流感疫苗的生產(chǎn)工藝亟待更新。其次,現(xiàn)有流感疫苗的有效成分是流感病毒HA和NA抗原。流感病毒具有易錯(cuò)傾向的RNA依賴性RNA聚合酶,使得HA和NA抗原不斷發(fā)生抗原漂移;流感病毒的宿主極為廣泛,可以感染鳥類、馬、豬等多種動(dòng)物并在這些動(dòng)物體內(nèi)發(fā)生基因節(jié)段的重組。因此,現(xiàn)有的季節(jié)性流感疫苗每年都需要更新。這不但需要對(duì)每年的病毒流行株進(jìn)行預(yù)測(cè),而且,每年接種流感疫苗,除了加重國(guó)家公共衛(wèi)生經(jīng)費(fèi)的負(fù)擔(dān)造成大量不必要的浪費(fèi)之外,還不可避免的在人體中引入大量外源核酸,對(duì)人體健康造成潛在的威脅。因此,更新現(xiàn)有流感疫苗生產(chǎn)工藝,研發(fā)新類型的流感疫苗成為流感疫苗研究領(lǐng)域的發(fā)展方向。 病毒樣顆粒(Virus-like particle, VLP)作為一種候選疫苗具有免疫原性好,沒有感染性,穩(wěn)定性好,不易失活等特點(diǎn),因此在病毒感染性疾病的疫苗研發(fā)中極具研究?jī)r(jià)值。國(guó)內(nèi)外已有大量關(guān)于病毒樣顆粒的研究報(bào)道,包括人類乳頭瘤病毒樣顆粒、人類免疫缺陷病毒樣顆粒、輪狀病毒樣顆粒等。其中,HPV-VLP疫苗已經(jīng)在歐洲和美國(guó)上市,展現(xiàn)出了VLP疫苗良好的應(yīng)用前景。由昆蟲細(xì)胞衍生的流感VLP是最有希望替代現(xiàn)有流感疫苗的候選疫苗。目前已經(jīng)成功重組的流感VLP包括H9N2、H3N2、H1N1、H5N1等多種亞型,這些VLP都具有免疫原性強(qiáng),保護(hù)效果好,易于構(gòu)建表達(dá)等優(yōu)點(diǎn)。因此,研制流感病毒樣顆粒疫苗具有極為重要的科學(xué)價(jià)值和社會(huì)效益。但是國(guó)內(nèi)關(guān)于流感病毒樣顆粒的文獻(xiàn)還比較少,國(guó)外也未見關(guān)于將流感病毒樣顆粒與現(xiàn)行裂解疫苗進(jìn)行比較的報(bào)道。 現(xiàn)有的大部分通用疫苗研究以人來(lái)源的流感病毒M2蛋白外功能區(qū)(M2e)序列作為靶點(diǎn)。然而,M2e免疫原性低,M2e氨基酸序列在人來(lái)源和禽來(lái)源的A型流感病毒之間存在5-6個(gè)氨基酸殘基的差異,且M2e誘導(dǎo)機(jī)體產(chǎn)生細(xì)胞免疫的能力較差。NP418-426表位是NP蛋白的免疫優(yōu)勢(shì)表位,與IFN-γ的產(chǎn)生高度相關(guān)并且能夠有效的誘導(dǎo)特異的CTL發(fā)生免疫應(yīng)答;禽流感病毒來(lái)源的NP418-426表位可以被人流感病毒特異的CTL交叉識(shí)別。因此,NP418-426表位有希望成為基于人來(lái)源M2e序列的流感病毒通用疫苗的重要組成成分,用以提高流感通用疫苗的種間交叉免疫保護(hù)效果,進(jìn)一步提高通用疫苗的廣譜性。乙肝核心蛋白(HBc)是使用最為廣泛的外源表位載體。HBc可在體內(nèi)或體外自組裝成病毒樣顆粒(VLP),將外源表位與HBc構(gòu)建融合蛋白可以有效的誘導(dǎo)T細(xì)胞和B細(xì)胞免疫反應(yīng)�；谝陨涎芯勘尘�,本研究分為兩部分: 第一部分甲型H1N1病毒樣顆粒疫苗的初步研究 1.通過昆蟲-桿狀病毒表達(dá)系統(tǒng)表達(dá)A/California/07/2009(H1N1)毒株的VLP。使用DEAE陰離子交換介質(zhì)、Sepharose 4FF凝膠介質(zhì)純化甲型H1N1 VLP。電鏡觀察可知,純化的甲型H1N1 VLP結(jié)構(gòu)完整,直徑在100nm左右。通過Western blot、血凝、單向免疫擴(kuò)散等實(shí)驗(yàn)進(jìn)一步證實(shí),獲得了重組甲型H1N1 VLP蛋白抗原并具有良好的免疫活性。 2.以10ug HA為免疫劑量,腹腔免疫4-6周齡雌性Balb/c小鼠,ELISA結(jié)果證實(shí),研制的甲型H1N1 VLP能夠誘導(dǎo)機(jī)體產(chǎn)生高滴度的特異性抗體,且甲型H1N1 VLP較甲型H1N1裂解疫苗能夠誘導(dǎo)機(jī)體產(chǎn)生更高滴度的IgG抗體。 3.選用A/Beijing/501/2009 (H1N1)甲型H1N1流行株進(jìn)行攻毒,結(jié)果顯示,甲型H1N1 VLP候選疫苗能夠保護(hù)小鼠抵抗50LD_(50)劑量的致死性攻擊,保護(hù)率達(dá)到100%。 結(jié)論:研制的甲型H1N1 VLP候選疫苗具有良好的免疫原性,能夠刺激機(jī)體產(chǎn)生體液免疫應(yīng)答和細(xì)胞免疫應(yīng)答,且生產(chǎn)較為便利。昆蟲細(xì)胞生產(chǎn)的流感病毒VLP是值得深入研究,以期有希望替代現(xiàn)有流感疫苗的候選疫苗之一。 第二部分甲型流感通用病毒樣顆粒疫苗的初步研究 1.使用大腸桿菌表達(dá)融合蛋白3M2e-NP-HBc、3M2e-HBc以及HBc N端149個(gè)氨基酸HBc-N149。采用His-trap純化介質(zhì)對(duì)重組蛋白進(jìn)行純化。電鏡觀察可知,純化的重組蛋白可形成大小均一,結(jié)構(gòu)完整的病毒樣顆粒。通過M2e單克隆抗體進(jìn)行Western blot實(shí)驗(yàn)可知,3M2e-NP-HBc及3M2e-HBc VLP抗原具有良好的M2e特異的免疫活性。 2.以20ug重組蛋白為免疫劑量,選取4-6周齡雌性Balb/c小鼠進(jìn)行動(dòng)物實(shí)驗(yàn)可知,重組3M2e-NP-HBc和3M2e-HBc VLP抗原均能夠誘導(dǎo)機(jī)體產(chǎn)生高滴度的M2e特異性抗體。3M2e-NP-HBc較3M2e-HBc能夠誘導(dǎo)機(jī)體產(chǎn)生更高滴度的IgG2a抗體且3M2e-NP-HBc較3M2e-HBc能夠刺激機(jī)體產(chǎn)生更多的IFN-γ分泌型淋巴細(xì)胞。 3.分別使用A/Beijing/501/2009 (H1N1)甲型H1N1毒株、A/PR/8/34(H1N1)及A/Ostrich/SuZhou/097/2003 (H5N1)高致病性禽流感毒株進(jìn)行攻毒實(shí)驗(yàn)。結(jié)果顯示,3M2e-NP-HBc和3 2e HBc均能夠保護(hù)小鼠抵抗10LD_(50)的A/Beiji g/501/2 09 (H1N1)、A/PR/8/34(H1N1)毒株的致死性攻擊,保護(hù)率達(dá)到100%。3M2e-NP-HBc較3M2e-HBc能夠更好的保護(hù)小鼠抵抗10LD_(50)的A/Ostrich/SuZhou/097/2003 (H5N1)毒株的致死性攻擊。 結(jié)論:NP418-426表位的嵌入能夠有效的誘導(dǎo)機(jī)體產(chǎn)生細(xì)胞免疫應(yīng)答,有效的提高基于M2e的甲型流感通用候選疫苗的種間交叉保護(hù)效果。流感病毒內(nèi)部蛋白作為潛在的通用疫苗候選抗原應(yīng)當(dāng)進(jìn)行深入的研究。
[Abstract]:As of July 18, 2010, influenza A (H1N1) outbreak in Mexico has killed 18,336 people in 214 countries worldwide; since 2003, the highly pathogenic H5N1 avian influenza virus has caused 310 deaths worldwide. Vaccines are the most effective means to prevent and control influenza. The development of influenza vaccines has gone through inactivated vaccines, lysed vaccines, live attenuated vaccines, subunit vaccines and so on. Although seasonal split influenza vaccines can produce effective protection in the population, there are still many problems. First, the traditional influenza vaccines The production process is dependent on chicken embryos, which is difficult to meet the demand for vaccines in large-scale influenza outbreaks, and egg albumin is susceptible to allergies. So far, there is no ideal influenza vaccine for the elderly, children and special populations. Therefore, the existing influenza vaccine production process needs to be updated urgently. Secondly, the effective ingredient of the existing influenza vaccine is influenza virus H. A and NA antigens. Influenza viruses have a RNA dependent RNA polymerase which is prone to be misaligned, which makes the antigen shift of HA and NA antigens. The host of influenza virus is very extensive, * it can infect many kinds of animals such as birds, horses, pigs and so on, and reorganize gene segments in these animals. New. This requires not only the annual prediction of the virus epidemic strains, but also the annual vaccination of influenza vaccines, in addition to increasing the burden of national public health expenditure caused by a large number of unnecessary waste, but also the inevitable introduction of a large number of exogenous nucleic acids in the human body, a potential threat to human health. The development of new types of influenza vaccine has become the development direction of influenza vaccine research.
Virus-like particles (VLP), as a candidate vaccine, have good immunogenicity, no infectivity, good stability, and are not easy to inactivate. Therefore, it is of great value in the research and development of vaccine for viral infectious diseases. Among them, HPV-VLP vaccines have been marketed in Europe and the United States, showing a promising future for VLP vaccines. Influenza VLP derived from insect cells is the most promising alternative to existing influenza vaccines. H5N1 and other subtypes, these VLPs have the advantages of strong immunogenicity, good protective effect, easy construction and expression. Therefore, the development of influenza-like granule vaccine has extremely important scientific value and social benefits. Comparison of split vaccine was reported.
However, M2e immunogenicity is low. There are 5-6 amino acid residues in the M2e amino acid sequence between human and avian influenza A viruses, and the ability of M2e to induce cellular immunity is poor. NP epitope is an immunodominant epitope of NP protein, which is highly correlated with the production of IFN-gamma and can effectively induce specific CTL immune response. NP418-426 epitope derived from avian influenza virus can be cross-identified by human influenza virus-specific CTL. Therefore, NP418-426 epitope is hopeful to become a universal influenza virus based on human M2e sequence. Hepatitis B core protein (HBc) is the most widely used exogenous epitope vector. HBc can self-assemble into virus-like particles (VLP) in vivo or in vitro, and construct fusion proteins between exogenous epitopes and HBc. In order to effectively induce T cell and B cell immune response, based on the above research background, the study is divided into two parts:
The first part is a preliminary study of a H1N1 virus like particle vaccine.
1. Expressing the VLP of A/California/07/2009 (H1N1) strain by insect-baculovirus expression system. Using DEAE anion exchange medium and Sepharose 4FF gel medium to purify A/H1N1 VLP. The structure of purified A/H1N1 VLP was intact and its diameter was about 100 nm. It was confirmed that the recombinant H1N1 VLP protein antigen was obtained and had good immunological activity.
2. Female Balb/c mice aged 4-6 weeks were immunized by intraperitoneal injection of 10ug HA. ELISA results showed that the developed A H1N1 VLP could induce high titer of specific antibodies, and A H1N1 VLP could induce higher titer of IgG antibodies than A H1N1 lysis vaccine.
3. A/Beijing/501/2009 (H1N1) influenza A H1N1 strain was selected to attack the virus. The results showed that the candidate vaccine of A H1N1 VLP could protect mice against lethal attack of 50 LD_ (50) dose, and the protection rate reached 100%.
CONCLUSION: The candidate vaccine for influenza A H1N1 VLP has good immunogenicity, can stimulate humoral and cellular immune responses, and is convenient for production.
The second part is a preliminary study of influenza A virus like particle vaccine.
1. The fusion protein 3M2e-NP-HBc, 3M2e-HBc and 149 amino acids HBc-N149 were expressed in E. coli. The recombinant protein was purified by his-trap purification medium. The purified recombinant protein could form virus-like particles with uniform size and complete structure. The Western blot test with M2e monoclonal antibody showed that the recombinant protein was 3. M2e-NP-HBc and 3M2e-HBc VLP antigen have good M2e specific immune activity.
2. Using 20ug recombinant protein as the immune dose, female Balb/c mice aged 4-6 weeks were selected for animal experiment. The results showed that recombinant 3M2e-NP-HBc and 3M2e-HBc VLP antigens could induce high titer of M2e-specific antibody. 3M2e-NP-HBc could induce higher titer of IgG2a antibody than 3M2e-HBc and 3M2e-NP-HBc could prick higher titer of IgG2a antibody than 3M2e-HBc. The stimulated organism produces more IFN- - ray secreting lymphocytes.
3. Attack experiments were carried out using A/Beijing/501/2009 (H1N1) A/H1N1 strain, A/PR/8/34 (H1N1) and A/Ostrich/SuZhou/097/2003 (H5N1) highly pathogenic avian influenza strains. The results showed that 3M2e-NP-HBc and 32e-HBc could protect mice against lethal attack by 10LD_ (50) A/Beiji g/501/209 (H1N1), A/PR/8/34 (H1N1) strains. Compared with 3M2e-HBc, 3M2e-NP-HBc could protect mice against lethal attack of 10LD_ (50) A/Ostrich/SuZhou/097/2003 (H5N1).
CONCLUSION: NP418-426 epitope insertion can effectively induce cellular immune response and improve the cross-protection effect of M2e-based universal vaccine candidates for influenza A. As a potential candidate antigen for universal vaccine, influenza virus internal protein should be further studied.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392
本文編號(hào)：2182618
[Abstract]:As of July 18, 2010, influenza A (H1N1) outbreak in Mexico has killed 18,336 people in 214 countries worldwide; since 2003, the highly pathogenic H5N1 avian influenza virus has caused 310 deaths worldwide. Vaccines are the most effective means to prevent and control influenza. The development of influenza vaccines has gone through inactivated vaccines, lysed vaccines, live attenuated vaccines, subunit vaccines and so on. Although seasonal split influenza vaccines can produce effective protection in the population, there are still many problems. First, the traditional influenza vaccines The production process is dependent on chicken embryos, which is difficult to meet the demand for vaccines in large-scale influenza outbreaks, and egg albumin is susceptible to allergies. So far, there is no ideal influenza vaccine for the elderly, children and special populations. Therefore, the existing influenza vaccine production process needs to be updated urgently. Secondly, the effective ingredient of the existing influenza vaccine is influenza virus H. A and NA antigens. Influenza viruses have a RNA dependent RNA polymerase which is prone to be misaligned, which makes the antigen shift of HA and NA antigens. The host of influenza virus is very extensive, * it can infect many kinds of animals such as birds, horses, pigs and so on, and reorganize gene segments in these animals. New. This requires not only the annual prediction of the virus epidemic strains, but also the annual vaccination of influenza vaccines, in addition to increasing the burden of national public health expenditure caused by a large number of unnecessary waste, but also the inevitable introduction of a large number of exogenous nucleic acids in the human body, a potential threat to human health. The development of new types of influenza vaccine has become the development direction of influenza vaccine research.
Virus-like particles (VLP), as a candidate vaccine, have good immunogenicity, no infectivity, good stability, and are not easy to inactivate. Therefore, it is of great value in the research and development of vaccine for viral infectious diseases. Among them, HPV-VLP vaccines have been marketed in Europe and the United States, showing a promising future for VLP vaccines. Influenza VLP derived from insect cells is the most promising alternative to existing influenza vaccines. H5N1 and other subtypes, these VLPs have the advantages of strong immunogenicity, good protective effect, easy construction and expression. Therefore, the development of influenza-like granule vaccine has extremely important scientific value and social benefits. Comparison of split vaccine was reported.
However, M2e immunogenicity is low. There are 5-6 amino acid residues in the M2e amino acid sequence between human and avian influenza A viruses, and the ability of M2e to induce cellular immunity is poor. NP epitope is an immunodominant epitope of NP protein, which is highly correlated with the production of IFN-gamma and can effectively induce specific CTL immune response. NP418-426 epitope derived from avian influenza virus can be cross-identified by human influenza virus-specific CTL. Therefore, NP418-426 epitope is hopeful to become a universal influenza virus based on human M2e sequence. Hepatitis B core protein (HBc) is the most widely used exogenous epitope vector. HBc can self-assemble into virus-like particles (VLP) in vivo or in vitro, and construct fusion proteins between exogenous epitopes and HBc. In order to effectively induce T cell and B cell immune response, based on the above research background, the study is divided into two parts:
The first part is a preliminary study of a H1N1 virus like particle vaccine.
1. Expressing the VLP of A/California/07/2009 (H1N1) strain by insect-baculovirus expression system. Using DEAE anion exchange medium and Sepharose 4FF gel medium to purify A/H1N1 VLP. The structure of purified A/H1N1 VLP was intact and its diameter was about 100 nm. It was confirmed that the recombinant H1N1 VLP protein antigen was obtained and had good immunological activity.
2. Female Balb/c mice aged 4-6 weeks were immunized by intraperitoneal injection of 10ug HA. ELISA results showed that the developed A H1N1 VLP could induce high titer of specific antibodies, and A H1N1 VLP could induce higher titer of IgG antibodies than A H1N1 lysis vaccine.
3. A/Beijing/501/2009 (H1N1) influenza A H1N1 strain was selected to attack the virus. The results showed that the candidate vaccine of A H1N1 VLP could protect mice against lethal attack of 50 LD_ (50) dose, and the protection rate reached 100%.
CONCLUSION: The candidate vaccine for influenza A H1N1 VLP has good immunogenicity, can stimulate humoral and cellular immune responses, and is convenient for production.
The second part is a preliminary study of influenza A virus like particle vaccine.
1. The fusion protein 3M2e-NP-HBc, 3M2e-HBc and 149 amino acids HBc-N149 were expressed in E. coli. The recombinant protein was purified by his-trap purification medium. The purified recombinant protein could form virus-like particles with uniform size and complete structure. The Western blot test with M2e monoclonal antibody showed that the recombinant protein was 3. M2e-NP-HBc and 3M2e-HBc VLP antigen have good M2e specific immune activity.
2. Using 20ug recombinant protein as the immune dose, female Balb/c mice aged 4-6 weeks were selected for animal experiment. The results showed that recombinant 3M2e-NP-HBc and 3M2e-HBc VLP antigens could induce high titer of M2e-specific antibody. 3M2e-NP-HBc could induce higher titer of IgG2a antibody than 3M2e-HBc and 3M2e-NP-HBc could prick higher titer of IgG2a antibody than 3M2e-HBc. The stimulated organism produces more IFN- - ray secreting lymphocytes.
3. Attack experiments were carried out using A/Beijing/501/2009 (H1N1) A/H1N1 strain, A/PR/8/34 (H1N1) and A/Ostrich/SuZhou/097/2003 (H5N1) highly pathogenic avian influenza strains. The results showed that 3M2e-NP-HBc and 32e-HBc could protect mice against lethal attack by 10LD_ (50) A/Beiji g/501/209 (H1N1), A/PR/8/34 (H1N1) strains. Compared with 3M2e-HBc, 3M2e-NP-HBc could protect mice against lethal attack of 10LD_ (50) A/Ostrich/SuZhou/097/2003 (H5N1).
CONCLUSION: NP418-426 epitope insertion can effectively induce cellular immune response and improve the cross-protection effect of M2e-based universal vaccine candidates for influenza A. As a potential candidate antigen for universal vaccine, influenza virus internal protein should be further studied.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前3條
1 吳小鋒;岳萬(wàn)福;劉劍梅;李廣立;繆云根;;Bac-to-Bac系統(tǒng)的研究進(jìn)展及在家蠶中的應(yīng)用[J];蠶業(yè)科學(xué);2007年01期
2 王惠明;吳玉章;;以病毒樣顆粒為平臺(tái)的分子載體和疫苗設(shè)計(jì)[J];免疫學(xué)雜志;2006年01期
3 姚志東;張良艷;劉坤;邢麗;王希良;;甲型H1N1流感病毒疫苗株高適應(yīng)性MDCK細(xì)胞的篩選及鑒定[J];免疫學(xué)雜志;2011年03期
，本文編號(hào)：2182618
本文鏈接：http://www.sikaile.net/xiyixuelunwen/2182618.html
最近更新
教材專著
